Phosphotriesterase from agrobacterium radiobacter P230

ABSTRACT

The present invention provides enzymes capable of hydrolysing organophosphate (OP) molecules. In particular, the invention provides a phosphotriesterase enzyme identified from an  Agrobacterium radiobacter  strain isolated from soil that hydrolyses OP pesticides, and the gene encoding that enzyme. The invention also provides mutants of the identified phosphotriesterase enzyme which have altered substrate specificity. The use of these enzymes in bioremediation strategies is also provided.

This application is a divisional of U.S. Ser. No. 10/477,469, now U.S. Pat. No. 8,293,244, issued Oct. 23, 2012, a §371 national stage of PCT International Application No. PCT/AU2002/00594, filed May 15, 2002, claiming priority of Australian Patent Application No. PR 5023, filed May 15, 2001, the contents of each of which are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to enzymes capable of hydrolyzing organophosphate (OP) molecules. In particular, the invention relates to a phosphotriesterase enzyme identified from an Agrobacterium radiobacter strain isolated from soil that hydrolyses OP pesticides, and the gene encoding that enzyme. The invention also relates to mutants of the identified phosphotriesterase enzyme which have altered substrate specificity.

BACKGROUND OF THE INVENTION

Residues of organophosphate insecticides are undesirable contaminants of the environment and a range of commodities. Areas of particular sensitivity include contamination of soil, irrigation tailwater that is re-cycled, used by irrigators downstream or simply allowed to run off-farm, and residues above permissible levels in agricultural and horticultural exports. Poisoning with organophosphates presents a problem for agricultural workers that are exposed to these chemicals, as well as military personnel exposed to organophosphates used in chemical warfare. Furthermore, the stockpiling of organophosphorus nerve agents has resulted in the need to detoxify these stocks. Bioremediation strategies are therefore required for eliminating or reducing these organophosphate residues and/or stockpiles.

One proposed strategy involves the use of enzymes capable of immobilising or degrading the organophosphate residues. Such enzymes may be employed, for example, in bioreactors through which contaminated water could be passed, or in washing solutions after post-harvest disinfestation of fruit, vegetables or animal products to reduce residue levels and withholding times. Suitable enzymes for degrading organophosphate residues include OP hydrolases from bacteria (Mulbry, 1992; Mulbry and Kearney, 1991; Cheng et al., 1999; U.S. Pat. No. 5,484,728; U.S. Pat. No. 5,589,386), vertebrates (Wang et al., 1993; 1998; Gan at al, 1991; Broomfield et al., 1999) and OP resistant insects (WO 95/19440 and WO 97/19176). It is desirable that the OP hydrolases degrade the organophosphate residues at a rapid rate.

The most thoroughly studied OP degrading enzyme is bacterial organophosphate dihydrolase (OPD), which is encoded by identical genes on dissimilar plasmids in both Flavobacterium sp. ATCC 27551 and Brevundimonas diminuta MG (Harper of al., 1988; Mulbry and Karns, 1989). OPD is a homodimeric protein that is capable of hydrolysing a wide range of phosphate triesters (both oxon and thion OPs) (Dumas et al., 1989a, b). Its reaction mechanism directly or indirectly involves metal ions, preferably Zn⁺⁺. OPD has no detectable activity with phosphate monoesters or diesters (Dumas et al., 1989a, b; 1990).

OPD homologues (phosphotriesterase homology proteins, or PHPs) have been identified in the genomes of Escherichia coli (ePHP), Mycobacterium tuberculosis (mtPHP) and Mycoplasma pneumoniae (mpPHP), although only ePHP has been tested for phosphotriesterase activity (Scanlan and Reid, 1995; Buchbinder et al., 1998). No activity was detected in ePHP crude lysates with any of the substrates tested, such as p-nitrophenyl acetate, bis(p-nitrophenyl) phosphate, paraoxon and p-nitrophenyl phosphate.

OPD homologues have also been identified in vertebrates (Davies et al., 1997), although their function in these organisms is unknown. OPD, ePHP, mtPHP and mammalian PHPs are 27-30% identical at the amino acid level, while mpPHP is less similar. Amino acid residues involved in Zn⁺⁺ binding are conserved across the six members of the phosphotriesterase family identified to date (Buchbinder et al., 1998).

Three other distinct OP hydrolysing enzymes have been isolated from bacteria with a history of exposure to OPs (Mulbry and Karns, 1989; Mulbry, 1992; Cheng et al., 1999). The two for which sequence data are available are unrelated to each other and to OPD. One, a prolidase from Alteromonas sp., normally functions in hydrolysis of X-Pro dipeptides. Its activity for insecticidal OPs is reported as modest, although it has not been reported in terms of k_(cat)/K_(m) specificity constants (Cheng et al., 1999). The other, an aryldialkylphosphatase (ADPase) from Nocardia sp. strain B-1, has a turnover number for ethyl parathion that is 4500-fold lower than that reported for OPD (Mulbry and Karns, 1989; Mulbry, 1992).

Paraoxonase, or PON1, is a distinct OP hydrolysing enzyme found in mammals. Like OPD it is a metalloenzyme, preferring Ca⁺⁺ in this case, which is associated with low density lipoproteins in plasma and normally involved in metabolism of oxidised lipid compounds (Gan et al., 1991; Sorenson et al., 1995). It has high activity for paraoxon, with a specificity constant of around 10⁶ M⁻¹sec⁻¹ (Doom et al., 1999; Hong and Raushel, 1999).

There is also evidence for other, so-called diisopropyl fluorophosphatase (DFPase) enzymes in a wide range of vertebrates, invertebrates and microorganisms (Wang et al., 1998; Hoskin et al., 1999; Billecke et al., 1999). These enzymes are notably diverse in many of their biochemical properties but are all characterised by their hydrolytic activity against OP chemical warfare agents. Limited sequence data suggest that they are unrelated to all the other OP hydrolytic enzymes described above.

OP resistant blowflies and houseflies have been the source of esterase enzymes with activity against oxon OPs like chlorfenvinfos (CVP) and carboxylester OPs like malathion (Newcomb et al., 1997; Campbell et al. 1998; Claudianos et al. 1999; WO 95/19440; WO 97/19176). A Gly to Asp substitution at residue 137 in blowfly esterase E3 (and its housefly ortholog, ALI) resulted in the acquisition of activity for CVP, while a Trp to Leu/Ser mutation at residue 251 in the same enzyme resulted in activity against malathion. However, the specificity constants of these enzymes for their OP substrates are orders of magnitude less than those of OPD for paraoxon.

There is a need for further OP degrading enzymes which can be used in bioremediation strategies.

SUMMARY OF THE INVENTION

The present inventors have developed a rapid and sensitive fluorimetric assay for coumaphos (a thion OP insecticide) hydrolysis and used it to isolate a bacterium from contaminated soil that is capable of using OPs as the sole phosphorus source. 16S rDNA sequencing identified the bacterium (isolate P230) as a strain of Agrobacterium radiobacter. The present inventors have also isolated and characterized the enzyme responsible for this coumaphos hydrolytic activity and provide methods for the use of this enzyme in bioremediation strategies.

In one aspect, the present invention provides a substantially purified polypeptide, the polypeptide being selected from:

-   -   (i) a polypeptide comprising a sequence provided in SEQ ID NO:1;     -   (ii) a polypeptide comprising a sequence provided in SEQ ID         NO:2;     -   (iii) a polypeptide comprising a sequence provided in SEQ ID         NO:3;     -   (iv) a polypeptide comprising a sequence provided in SEQ ID         NO:4; or     -   (v) a polypeptide comprising a sequence which is greater than         90% identical to any one of (i) to (iv),         wherein the polypeptide is capable of hydrolysing an         organophosphate molecule.

Preferred organophosphate molecules include, but are not limited to, coumaphos, coroxon, paraoxon, parathion, parathion-methyl, phosmet, fenthion, diazinon, chlorpyrifos, dMUP, DFP, dimethoate, malathion, and malaoxon. More preferably, the organophosphate is phosmet or fenthion.

In a preferred embodiment, the polypeptide can be purified from an Agrobacterium sp.

In a further preferred embodiment, the polypeptide is at least 95% identical to any one of (i) to (iv), more preferably at least 97% identical, and even more preferably at least 99% identical to any one of (i) to (iv).

In another aspect, the present invention provides a substantially purified polypeptide, the polypeptide being selected from:

-   -   (i) a polypeptide comprising the sequence provided in SEQ ID         NO:1;     -   (ii) a polypeptide comprising the sequence provided in SEQ ID         NO:2; or     -   (iii) a polypeptide which is greater than 90% identical to (i)         or (ii).

In another aspect, a fusion polypeptide is provided which comprises a polypeptide according to the present invention fused to at least one other polypeptide sequence.

Preferably, the at least one other polypeptide is selected from the group consisting of: a polypeptide that enhances the stability of the polypeptide of the invention, and a polypeptide that assists in the purification of the fusion polypeptide.

Preferably, the at least one other polypeptide is the maltose-binding protein.

In another aspect, the present invention provides an isolated polynucleotide, the polynucleotide comprising a sequence selected from:

-   -   (i) a sequence of nucleotides shown in SEQ ID NO:5;     -   (ii) a sequence of nucleotides shown in SEQ ID NO:6;     -   (iii) a sequence of nucleotides shown in SEQ ID NO:7;     -   (iv) a sequence of nucleotides shown in SEQ ID NO:8;     -   (v) a sequence encoding a polypeptide according to the present         invention; or     -   (vi) a sequence which is at least 90% identical to any one         of (i) to (v),         wherein the polynucleotide encodes a polypeptide capable of         hydrolysing an organophosphate molecule.

Preferably, the polynucleotide is at least 95% identical, more preferably at least 97% identical, and even more preferably at least 99% identical to any one of (i) to (v).

In a further aspect, a vector is provided which comprises a polynucleotide according to the invention.

Preferably, the vector is suitable for the replication and/or expression of a polynucleotide. The vectors may be, for example, a plasmid, virus or phage vector provided with an origin of replication, and preferably a promotor for the expression of the polynucleotide and optionally a regulator of the promotor. The vector may contain one or more selectable markers, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian expression vector. The vector may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.

In another aspect, a host cell is provided which comprises a vector according to the invention.

In a further aspect, the present invention provides a process for preparing a polypeptide of the invention, the process comprising cultivating a host cell of the invention under conditions which allow production of the polypeptide, and recovering the polypeptide. Such cells can be used for the production of commercially useful quantities of the encoded polypeptide.

In another aspect, the present invention provides a composition for hydrolysing an organophosphate molecule, the composition comprising a polypeptide according to the invention, and one or more acceptable carriers.

In another aspect, the present invention provides a composition for hydrolysing an organophosphate molecule, the composition comprising a host cell of the invention, and one or more acceptable carriers.

It will be appreciated that the present invention can be used to hydrolyse organophosphates in a sample. For instance, after a crop has been sprayed with an organophosphate pesticide, the organophosphate residue can be hydrolysed from seeds, fruits and vegetables before human consumption. Similarly, organophosphate contaminated soil or water can be treated with a polypeptide of the invention.

Accordingly, in a further aspect the present invention provides a method for hydrolysing an organophosphate molecule in a sample, the method comprising exposing the sample to a polypeptide according to the invention.

Preferably, the polypeptide is provided as a composition of the invention.

Further, it is preferred that the method further comprises exposing the sample to a divalent cation. Preferably, the divalent cation is zinc.

Preferably, the sample is selected from the group consisting of; soil, water, biological material, or a combination thereof. Preferred biological samples include matter derived from plants such as seeds, vegetables or fruits, as well as matter derived from animals such as meat.

Preferred organophosphate molecules include, but are not limited to, coumaphos, coroxon, paraoxon, parathion, parathion-methyl, phosmet, fenthion, diazinon, chlorpyrifos, dMUP, DFP, dimethoate, malathion, and malaoxon. More preferably, the organophosphate is phosmet or fenthion.

The sample can be exposed to the polypeptide via any available avenue. This includes providing the polypeptide directly to the sample, with or without carriers or excipients etc. The polypeptide can also be provided in the form of a host cell, typically a microorganism such as a bacterium or a fungus, which expresses a polynucleotide encoding the polypeptide of the invention. Usually, the polypeptide will be provided as a composition of the invention.

Organophosphate molecules in a sample can also be hydrolysed by exposing the sample to a transgenic plant which produces a polypeptide of the present invention.

Thus, in a further aspect a transgenic plant is provided which produces a polypeptide according to the invention.

In a further aspect, the present invention provides a method for hydrolysing an organophosphate molecule in a sample, the method comprising exposing the sample to a transgenic plant according to the invention.

Preferably, the sample is soil.

Further, it is preferred that the polypeptide is at least produced in the roots of the transgenic plant.

In yet another aspect, the present invention provides an isolated strain of Agrobacterium radiobacter deposited under NM01/21112 on 20 Apr. 2001 at Australian Government Analytical Laboratories.

In another aspect, the present invention provides a composition for hydrolysing an organophosphate molecule, the composition comprising the Agrobacterium radiobacter strain of the invention, and one or more acceptable carriers.

In yet another aspect, the present invention provides a method for hydrolysing an organophosphate molecule in a sample, the method comprising exposing the sample to an Agrobacterium radiobacter strain according to the invention.

The disclosure of the present invention can readily be used to isolate other bacterial species/strains which hydrolyse organophosphates. For example, other bacterial species/strains may be isolated using a fluormetric screening method as disclosed herein. Alternatively, probes and/or primers can be designed based on the polynucleotides of the present invention to identify bacteria which produce naturally occurring variants of the polypeptides of the present invention.

Accordingly, in a further aspect the present invention provides an isolated bacterium which produces a polypeptide according to the invention.

Preferably, the bacterium is an Agrobacterium sp. More preferably, the bacterium is a strain of Agrobacterium radiobacter.

In a further aspect, the present invention provides the use of an isolated naturally occurring bacterium which produces a polypeptide according to the invention for hydrolysing an organophosphate in a sample.

In a further aspect, the present invention provides a polymeric sponge or foam for hydrolysing an organophosphate molecule, the foam or sponge comprising a polypeptide according to the invention immobilized on a polymeric porous support.

Preferably, the porous support comprises polyurethane.

In a preferred embodiment, the sponge or foam further comprises carbon embedded or integrated on or in the porous support.

In a further aspect, the present invention provides a method for hydrolysing an organophosphate molecule in a sample, the method comprising exposing the sample to a sponge or foam according to the invention.

In another aspect, the present invention provides a biosensor for detecting the presence of an organophosphate, the biosensor comprising a polypeptide of the invention, and a means for detecting hydrolysis of an organophosphate molecule by the polypeptide.

In yet another aspect, the present invention provides a method for screening for agents which hydrolyse an organophosphate molecule, the method comprising

-   -   (i) exposing the organophosphate to a candidate agent, and     -   (ii) measuring a fluorescent signal produced from step (i),         wherein the fluorescent signal is indicative of hydrolysis of         the organophosphate.

Preferably, the organophosphate is coumaphos or coroxon.

Further, it is preferred that the agent is a polypeptide or a micro-organism.

The polypeptide of the present invention can be mutated, and the resulting mutants screened for altered activity such as changes in substrate specificity.

Thus, in a further aspect, the present invention provides a method of producing a polypeptide with enhanced ability to hydrolyse an organophosphate or altered substrate specificity for an organophosphate, the method comprising

-   -   i) mutating one or more amino acids of a first polypeptide         according to the present invention,     -   ii) determining the ability of the mutant to hydrolyse an         organophosphate, and     -   iii) selecting a mutant with enhanced ability to hydrolyse the         organophosphate or altered substrate specificity for the         organophosphate, when compared to the first polypeptide.

As outlined in the Example section, this method has been successfully applied to produce the polypeptides provided as SEQ ID NO:2 and SEQ ID NO:3.

Preferably, the first polypeptide is selected from any one if SEQ ID NO's: 1 to 4.

In a further aspect, the present invention provides a polypeptide produced according to the above method.

Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The invention will hereinafter be described by way of the following non-limiting Figures and Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Structure of coumaphos and its hydrolysis products.

FIG. 2: The DNA sequence of opdA (SEQ ID NO:5). The region encoding the signal peptide domain is given in bold, with the remaining sequence being referred to herein as SEQ ID NO:6.

FIG. 3: Amino acid sequence of OpdA (SEQ ID NO:1). The signal peptide is given in bold.

FIG. 4: Amino acid sequence alignment of OPD (SEQ ID NO:17) and OpdA. The secretion signals are given in bold.

KEY TO THE SEQUENCE LISTING

-   SEQ ID NO: 1—Polypeptide sequence of OpdA. -   SEQ ID NO: 2—Polypeptide sequence of OpdA minus the signal sequence. -   SEQ ID NO: 3—Polypeptide sequence of OpdA1. -   SEQ ID NO: 4—Polypeptide sequence of OpdA2. -   SEQ ID NO: 5—Polynucleotide sequence encoding OpdA. -   SEQ ID NO: 6—Polynucleotide sequence encoding OpdA minus the signal     sequence. -   SEQ ID NO: 7—Polynucleotide sequence encoding OpdA1. -   SEQ ID NO: 8—Polynucleotide sequence encoding OpdA2. -   SEQ ID NO's: 9 to 16—PCR primers. -   SEQ ID NO: 17—Polypeptide sequence of OPD from Flavobacterium sp.

DETAILED DESCRIPTION OF THE INVENTION

General Techniques

Unless otherwise indicated, the recombinant DNA techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (Editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present).

Organophosphates

Organophosphates are synthetic organophosphorus esters and related compounds such as phosphoroamidates. They have the general formula (RR′X)P═O or (RR′X)P═S, where R and R′ are short-chain groups. For insecticidal organophosphates X is a good leaving group, which is a requirement for the irreversible inhibition of acetylcholinesterase.

The polypeptides of the present invention hydrolyse the phosphoester bonds of organophosphates. These organophosphates can be, but are not limited to, oxon and thion OPs. The organophosphate can have aromatic or aliphatic leaving groups (X).

Although well known for their use as pesticides, organophosphates have also been used as nerve gases against mammals. Accordingly, it is envisaged that the polypeptides of the present invention will also be useful for hydrolysis of organophosphates which are not pesticides.

Polypeptides

By “substantially purified polypeptide” we mean a polypeptide that has generally been separated from the lipids, nucleic acids, other polypeptides, and other contaminating molecules with which it is associated in its native state. Preferably, the substantially purified polypeptide is at least 60% free, more preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.

The % identity of a polypeptide is determined by FASTA (Pearson and Lipman, 1988) analysis (GCG program) using the default settings and a query sequence of at least 50 amino acids in length, and whereby the FASTA analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the FASTA analysis aligns the two sequences over a region of at least 100 amino acids. More preferably, the FASTA analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the FASTA analysis aligns the two sequences over a region of at least 350 amino acids.

Amino acid sequence mutants of the polypeptides of the present invention can be prepared by introducing appropriate nucleotide changes into a nucleic acid sequence, or by in vitro synthesis of the desired polypeptide. Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final protein product possesses the desired characteristics. Examples of mutants of the present invention are provided in Example 8.

In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 30 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include sites identified as the active site(s). Other sites of interest are those in which particular residues obtained from various species are identical. These positions may be important for biological activity. These sites, especially those falling within a sequence of at least three other identically conserved sites, are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading of “exemplary substitutions”.

Since the sequence of SEQ ID NO:1 is 90% identical to that of the Flavobacterium OPD enzyme it is possible that SEQ ID NO:1 could be used to design mutants of the Flavobacterium OPD enzyme which have the desired activity but are less than 90% identical. More specifically, those amino acids important for hydrolysing an organophosphate molecule could be changed to match the polypeptides of the present invention and other amino acids not affecting this activity could also be changed to ensure the identity levels do not exceed 90%. Examples of such OPD mutants include the amino acid changes L272F and/or H257Y. Such mutants are also included in the present invention.

TABLE 1 Exemplary substitutions Original Exemplary Residue Substitutions Ala (A) val; leu; ile; gly Arg (R) lys Asn (N) gln; his; Asp (D) glu Cys (C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pro, ala His (H) asn; gln Ile (I) leu; val; ala Leu (L) ile; val; met; ala; phe Lys (K) arg Met (M) leu; phe; Phe (F) leu; val; ala Pro (P) gly Ser (S) thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; leu; met; phe, ala

Furthermore, if desired, unnatural amino acids or chemical amino acid analogues can be introduced as a substitution or addition into the polypeptide of the present invention. Such amino acids include, but are not limited to, the D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogues in general.

Also included within the scope of the invention are polypeptides of the present invention which are differentially modified during or after synthesis, e.g., by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. These modifications may serve to increase the stability and/or bioactivity of the polypeptide of the invention.

Polypeptides of the present invention can be produced in a variety of ways, including production and recovery of natural proteins, production and recovery of recombinant proteins, and chemical synthesis of the proteins. In one embodiment, an isolated polypeptide of the present invention is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, and recovering the polypeptide. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An effective medium refers to any medium in which a cell is cultured to produce a polypeptide of the present invention. Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.

Polynucleotides

By “isolated polynucleotide” we mean a polynucleotide which have generally been separated from the polynucleotide sequences with which it is associated or linked in its native state. Preferably, the isolated polynucleotide is at least 60% free, more preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated. Furthermore, the term “polynucleotide” is used interchangeably herein with the term “nucleic acid molecule”.

Polynucleotides of the present invention may possess one or more mutations when compared to SEQ ID NO's: 5 to 8. These mutations can be deletions, insertions, or substitutions of nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis on the nucleic acid). It is thus apparent that polynucleotides of the invention can be either naturally occurring or recombinant.

The % identity of a polynucleotide is determined by FASTA (Pearson and Lipman, 1988) analysis (GCG program) using the default settings and a query sequence of at least 150 nucleotides in length, and whereby the FASTA analysis aligns the two sequences over a region of at least 150 nucleotides. More preferably, the FASTA analysis aligns the two sequences over a region of at least 300 nucleotides. Even more preferably, the FASTA analysis aligns the two sequences over a region of at least 1050 nucleotides.

Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either. The minimum size of such oligonucleotides is the size required for the formation of a stable hybrid between an oligonucleotide and a complementary sequence on a nucleic acid molecule of the present invention. The present invention includes oligonucleotides that can be used as, for example, probes to identify nucleic acid molecules or primers to produce nucleic acid molecules.

Oligonucleotides and/or polynucleotides of the present invention may selectively hybridise to the sequences set out in SEQ ID NO's: 5 to 8 under high stringency. As used herein, stringent conditions are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% NaDodSO₄ at 50° C.; (2) employ during hybridisation a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfate at 42° C. in 0.2×SSC and 0.1% SDS.

Vectors

One embodiment of the present invention includes a recombinant vector, which includes at least one isolated nucleic acid molecule of the present invention, inserted into any vector capable of delivering the nucleic acid molecule into a host cell. Such a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention and that preferably are derived from a species other than the species from which the nucleic acid molecule(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid.

One type of recombinant vector comprises a nucleic acid molecule of the present invention operatively linked to an expression vector. The phrase operatively linked refers to the insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and effecting expression of a specified nucleic acid molecule. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, endoparasite, arthropod, other animal, and plant cells. Preferred expression vectors of the present invention can direct gene expression in bacterial, yeast, plant and mammalian cells.

Expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention. In particular, recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the host cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which function in bacterial, yeast, plant and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda, bacteriophage T7, T7lac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcohol oxidase, aiphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells. Additional suitable transcription control sequences include tissue-specific promoters and enhancers.

Recombinant molecules of the present invention may also (a) contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed polypeptide of the present invention to be secreted from the cell that produces the polypeptide and/or (b) contain fusion sequences which lead to the expression of nucleic acid molecules of the present invention as fusion proteins. Examples of suitable signal segments include any signal segment capable of directing the secretion of a protein of the present invention. Preferred signal segments include, but are not limited to, tissue plasminogen activator (t-PA), interferon, interleukin, growth hormone, histocompatibility and viral envelope glycoprotein signal segments, as well as natural signal sequences. In addition, a nucleic acid molecule of the present invention can be joined to a fusion segment that directs the encoded protein to the proteosome, such as a ubiquitin fusion segment. Recombinant molecules may also include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequences of nucleic acid molecules of the present invention.

Host Cells

Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present invention. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.

Suitable host cells to transform include any cell that can be transformed with a polynucleotide of the present invention. Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule (e.g., nucleic acid molecules encoding one or more proteins of the present invention). Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing proteins of the present invention or can be capable of producing such proteins after being transformed with at least one nucleic acid molecule of the present invention. Host cells of the present invention can be any cell capable of producing at least one protein of the present invention, and include bacterial, fungal (including yeast), parasite, arthropod, animal and plant cells. Preferred host cells include bacterial, mycobacterial, yeast, plant and mammalian cells. More preferred host cells include Agrobacterium, Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells (normal dog kidney cell line for canine herpesvirus cultivation), CRFK cells (normal cat kidney cell line for feline herpesvirus cultivation), CV-1 cells (African monkey kidney cell line used, for example, to culture raccoon poxvirus), COS (e.g., COS-7) cells, and Vero cells. Particularly preferred host cells are E. coli, including E. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains; Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCK cells; CRFK cells; CV-1 cells; COS cells; Vero cells; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246). Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH/3T3 cells, LMTK cells and/or HeLa cells.

Recombinant DNA technologies can be used to improve expression of transformed polynucleotide molecules by manipulating, for example, the number of copies of the polynucleotide molecules within a host cell, the efficiency with which those polynucleotide molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of polynucleotide molecules of the present invention include, but are not limited to, operatively linking polynucleotide molecules to high-copy number plasmids, integration of the polynucleotide molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of polynucleotide molecules of the present invention to correspond to the codon usage of the host cell, and the deletion of sequences that destabilize transcripts. The activity of an expressed recombinant protein of the present invention may be improved by fragmenting, modifying, or derivatizing polynucleotide molecules encoding such a protein.

Transgenic Plants

As generally described in WO 99/53037, the levels of organophosphates in a sample can be reduced by exposing the sample to a transgenic plant expressing a suitable enzyme. Typically, the sample is soil. Accordingly, the polynucleotide of the present invention can be expressed in a transgenic plant, particularly the roots of the plant, for hydrolysing organophosphate molecules in the sample.

The term “plant” refers to whole plants, plant organs (e.g. leaves, stems roots, etc), seeds, plant cells and the like. Plants contemplated for use in the practice of the present invention include both monocotyledons and dicotyledons. Exemplary dicotyledons include cotton, corn, tomato, tobacco, potato, bean, soybean, and the like.

Transgenic plants, as defined in the context of the present invention include plants (as well as parts and cells of said plants) and their progeny which have been genetically modified using recombinant DNA techniques to cause or enhance production of at least one protein of the present invention in the desired plant or plant organ.

The polypeptide of the present invention may be expressed constitutively in the transgenic plants during all stages of development. Depending on the use of the plant or plant organs, the proteins may be expressed in a stage-specific manner. Furthermore, depending on the use, the proteins may be expressed tissue-specifically.

The choice of the plant species is determined by the intended use of the plant or parts thereof and the amenability of the plant species to transformation.

Regulatory sequences which are known or are found to cause expression of a gene encoding a protein of interest in plants may be used in the present invention. The choice of the regulatory sequences used depends on the target plant and/or target organ of interest. Such regulatory sequences may be obtained from plants or plant viruses, or may be chemically synthesized. Such regulatory sequences are well known to those skilled in the art.

Other regulatory sequences such as terminator sequences and polyadenylation signals include any such sequence functioning as such in plants, the choice of which would be obvious to the skilled addressee. An example of such sequences is the 3′ flanking region of the nopaline synthase (nos) gene of Agrobacterium tumefaciens.

Several techniques are available for the introduction of the expression construct containing a DNA sequence encoding a protein of interest into the target plants. Such techniques include but are not limited to transformation of protoplasts using the calcium/polyethylene glycol method, electroporation and microinjection or (coated) particle bombardment. In addition to these so-called direct DNA transformation methods, transformation systems involving vectors are widely available, such as viral and bacterial vectors (e.g. from the genus Agrobacterium). After selection and/or screening, the protoplasts, cells or plant parts that have been transformed can be regenerated into whole plants, using methods known in the art. The choice of the transformation and/or regeneration techniques is not critical for this invention.

Compositions

Compositions of the present invention include excipients, also referred to herein as “acceptable carriers”. An excipient can be any material that the animal, plant, plant or animal material, or environment (including soil and water samples) to be treated can tolerate. Examples of such excipients include water, saline, Ringers solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal or o-cresol, formalin and benzyl alcohol. Excipients can also be used to increase the half-life of a composition, for example, but are not limited to, polymeric controlled release vehicles, biodegradable implants, liposomes, bacteria, viruses, other cells, oils, esters, and glycols.

Furthermore, the polypeptide of the present invention can be provided in a composition which enhances the rate and/or degree of organophosphate hydrolysis, or increases the stability of the polypeptide. For example, the polypeptide can be immobilized on a polyurethane matrix (Gordon et al., 1999), or encapsulated in appropriate liposomes (Petrikovics et al. 2000a and b). The polypeptide can also be incorporated into a composition comprising a foam such as those used routinely in fire-fighting (LeJeune et al., 1998). As would be appreciated by the skilled addressee, the polypeptide of the present invention could readily be used in a sponge or foam as disclosed in WO 00/64539, the contents of which are incorporated herein in their entirety.

One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal, plant, animal or plant material, or the environment (including soil and water samples). As used herein, a controlled release formulation comprises a composition of the present invention in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems. Preferred controlled release formulations are biodegradable (i.e., bioerodible).

A preferred controlled release formulation of the present invention is capable of releasing a composition of the present invention into soil or water which is in an area sprayed with an organophosphate pesticide. The formulation is preferably released over a period of time ranging from about 1 to about 12 months. A preferred controlled release formulation of the present invention is capable of effecting a treatment preferably for at least about 1 month, more preferably for at least about 3 months, even more preferably for at least about 6 months, even more preferably for at least about 9 months, and even more preferably for at least about 12 months.

The concentration of the polypeptide, vector, or host cell of the present invention that will be required to produce effective compositions for hydrolysing an organophosphate will depend on the nature of the sample to be decontaminated, the concentration of the organophosphate in the sample, and the formulation of the composition. The effective concentration of the polypeptide, vector, or host cell within the composition can readily be determined experimentally, as will be understood by the skilled artisan.

Biosensors

Biosensors are analytical devices typically consisting of a biologically active material such as an enzyme and a transducer that converts a biochemical reaction into a quantifiable electronic signal that can be processed, transmitted, and measured. A general review of biosensors which have been used for the detection of orangophosphorus compounds is provided by Rekha et al. (2000), the entire contents of which are incorporated by reference. The polypeptide of the present invention can be adapted for use in such biosensors.

EXAMPLES Example 1 Enriching Soil Samples for Microorganisms with Coumaphos Hydrolytic Activity

Fluorimetric Assay for Coumaphos Hydrolysis

Phosphotriesterase enzymes catalyse the cleavage of a phosphoester bond in organophosphate (OP) molecules to yield a phosphodiester and an alcohol: In the case of coumaphos (3-chloro-4-methyl-7-coumarinyl diethyl phosphorothioate), phosphotriesterase hydrolysis yields diethylthiophosphate and the fluorescent alcohol, chlorferon (3-chloro-7-hydroxy-4-methyl coumarin; FIG. 1). Coumaphos hydrolysis can therefore be measured fluorimetrically by the production of chlorferon, as measured by excitation at a wavelength of 355 nm and an emission intensity of 460 nm. Chlorferon fluorescence was linear over the range 0.01 μM to 2.5 μM.

All fluorescence measurements were performed in a POLARstar fluorimeter (BMG Technologies Pty Ltd, Australia) using 96-well white microtitre plates (FluoroNunc plates with PolySorp surface, Nalge Nunc International) and final reaction volumes of 100 μl. Stock solutions of coumaphos and chlorferon (0.4 mM) were prepared in 20% methanol. Crude assays of whole cells were performed in 100 μM coumaphos, 0.5% Triton X-100 and 50 mM Tris-HCl, pH8.0. Coumaphos hydrolytic assays of cell lysates were performed without the Triton X-100.

The fluorescence of bacterial colonies and stained polyacrylamide gels was examined using a hand-held long wavelength (approximately 340 nm) UV light (Gelman Sciences).

Enrichment Cultures

The phosphodiester hydrolysis products of phosphotriesterases can be used as phosphorus sources by a wide range of bacteria (Cook et al., 1978; Rosenberg and Alexander, 1979). An enrichment culture was therefore established in which 1 g of soil obtained from a domestic yard, which had previously been exposed to diazinon (a diethyl thion OP), served as an inoculum for 50 ml enrichment medium (Table 2), in which coumaphos was the only added phosphorus source.

TABLE 2 Composition of coumaphos enrichment media. Medium (per liter) Trace element solution (per liter) Tris 6.05 g (NH₄)₆Mo₇O₂₄•4H₂O 20 mg NH₄Cl 1 g H₃BO₃ 50 mg FeCl₂ 20 μg ZnCl₂ 30 mg KCl 0.5 g CoCl₂•6H₂O  3 mg Sodium acetate 0.68 g MnCl₂•4H₂O 10 mg MgSO₄ 0.1 g Cupric acetate 10 mg p-aminobenzoate 0.9 mg nicotinic acid 0.9 mg Trace element solution 10 ml Coumaphos 100 mM pH 7.0

Ten percent of the enrichment culture was subcultured twice into 50 ml fresh enrichment medium containing coumaphos as the sole phosphorus source. Coumaphos was then replaced with diazinon (at 100 mM) in the enrichment medium and the culture subcultured as before. After 3 days incubation at 28° C., the enrichment culture was further subcultured into media in which 100 mM parathion (another diethyl thion OP) was the sole phosphorus source. It was noted that after two days the culture had turned yellow (presumably due to the production of p-nitrophenol). This culture was then diluted in phosphorus-free medium and plated onto low salt LB plates (10 g/l tryptone, 5 g/l yeast extract and 2.5 g/l NaCl). After three days of growth at 28° C., approximately 100 colonies were picked randomly, re-streaked to ensure purity and then assayed for coumaphos hydrolytic activity using the microtitre plate assay described above. Fluorescence was measured after 8 hours at room temperature. One isolate (designated P230) demonstrated significant fluorescence and this isolate was examined further. Colonies of this isolate also demonstrated fluorescence on an agar plate containing coumaphos.

Example 2 Identification of Isolate P230

Isolate P230 was a Gram negative, catalase positive and oxidase positive, rod-shaped bacterium. To determine the identity of isolate P230, sequence analysis of the 16S rRNA gene was performed. DNA was extracted from isolate P230 according to the method of Rainey et al. (1992). Cells of a P230 culture that had been grown in low salt LB medium (2 ml) overnight at 28° C. were pelleted by centrifugation in a microfuge (12 000 rpm/2 minutes). The cell pellet was resuspended in 400 μl STE buffer (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, pH8.0) and 5 μl of a freshly-prepared lysozyme solution (0.3 μg/μl) was added. After incubation at 37° C. for 20 minutes, Proteinase K (15 μl of a 1% solution) and SDS (10 μl of a 25% solution) were added and the reactions incubated at 60° C. for 30 minutes. DNA preparations were then extracted sequentially with an equal volume of buffer-saturated phenol, and then an equal volume of chloroform.

The 16S rRNA gene was amplified from the extracted DNA by PCR using bacterial universal primers 27f (5′ AGAGTTTGATCMTGGCTCAG 3′) (SEQ ID NO: 9) and 1492r (5′ TACGGYTACCTTGTTACGACTT 3′) (SEQ ID NO: 10), the names of which are based on the numbering system of the E. coli 16S rRNA gene (Lane, 1991). Approximately 1320 bp of the 16S rRNA gene from isolate P230 was obtained and sequence similarities were performed using the FASTA algorithm (Pearson and Lipman, 1988). The 16S rRNA gene of isolate P230 was very similar in sequence to that of other Agrobacterium strains (Table 3).

TABLE 3 Nucleic acid sequence comparisons of the 16S rRNA genes of isolate P230 with those of various Agrobacterium strains. Agrobacterium strains Sequence Identity (%) Agrobacterium radiobacter LMG383 100 Agrobacterium sp LMG11936 99.7 Agrobacterium sp. MSMC211 99.5 Agrobacterium sp. LMG11915 99.3

These results suggested that isolate P230 was an Agrobacterium strain. The utilization of carbon sources by isolate P230 was examined using the Biolog system (Oxoid), according to procedures recommended by the manufacturer. The carbon utilization profile was then compared with that of known species of Agrobacterium (Table 4; Krieg and Holt, 1984). The isolate was capable of using sucrose, ornithine and glucose as carbon sources. This, along with a positive oxidase reaction determined using Kovac's method (Kovac, 1956), suggested that the isolate was most similar to either A. tumefaciens biovar1 or A. radiobacter biovar1.

TABLE 4 Carbon utilization profiles and oxidase status of isolate P230 and known Agrobacterium species. Agrobacterium spp. Carbon A. A. A. A. A. source/ tumefaciens tumefaciens radiobacter radiobacter rhizogenes Isolate test biovar1 biovar2 biovar1 biovar2 biovar2 A. rubi P230 Tween 80 − − − − − − − Sucrose + − + − − − + Ornithine + + + + + + + D-glucose + + + + + + + Oxidase + − + − − − + test (Kovac)

A. tumefaciens biovar1 and A. radiobacter biovar1 can be distinguished by the presence of a tumour-inducing plasmid in the former. The tumour-inducing ability of strain P230 was tested in a tomato seedling by transferring a heavy suspension of bacteria in water to the leaves and using a sterile needle to pierce the surface of the leaves through the suspension. No evidence of tumours was seen after a period of four weeks. A. tumefaciens C58 was used as a positive control and produced tumours in this period of time. A cured strain of A. tumefaciens C58 was used as a negative control and produced the same effects in the test plant as isolate P230. Therefore isolate P230 was designated as a strain of Agrobacterium radiobacter biovar1.

Example 3 Constitutive Expression of Coumaphos Hydrolytic Activity

In order to determine if the phosphotriesterase activity of A. radiobacter P230 was constitutively expressed, regardless of the presence of OPs, the parathion hydrolytic activities of cultures of A. radiobacter P230 were examined in the presence and absence of parathion (Table 5). Growth was monitored by measuring the optical density of the cultures at 595 nm in a BioRad Model 3550-UV microplate spectrophotometer. Parathion hydrolytic activity was assayed according to the procedure of Serdar et al. (1989). This involved measuring the formation of p-nitrophenol from parathion at 405 nm in a BioRad Model 3550-UV microplate spectrophotometer. The reaction mixture contained 880 μM parathion in 50 mM Tris-HCl pH8.0 (this reaction also contained 5% methanol). Table 5 shows that parathion hydrolytic activity was constitutively expressed in isolate P230 and that the majority of this activity was expressed in early- to mid-log phase.

Example 4 Native Polyacrylamide Gel Electrophoresis (PAGE) of P230 Extracts

To demonstrate that a single enzyme was involved in coumaphos hydrolysis, native gels of A. radiobacter P230 cell extracts were stained for coumaphos hydrolytic activity. A culture (50 ml) of A. radiobacter P230 in low salt LB broth was pelleted by centrifugation at 8000 g for 15 minutes and the cell pellet resuspended in 2 ml 50 mM Tris-HCl pH8.0. Cells were disrupted by sonication (five 15 second bursts at 4° C.) and large cell debris or intact cells removed by centrifugation (8000 g for 15 minutes). An aliquot of the resultant supernatant (containing 5 μg protein) was then separated on a 10% (29:1 acrylamide:bis) SDS-PAGE gel. Prior to loading, neither SDS nor β-mercaptoethanol was added to the sample and furthermore, the sample was not boiled as in conventional SDS-PAGE. After electrophoresis the gel was equilibrated for 5 min in 50 mM Tris-HCl pH8.0, and then incubated for a further 5 min in 50 mM Tris-HCl pH8.0, containing 8 μM coumaphos. The gel was then examined under UV light as described above. A major fluorescent band was detected, indicating that the P230 isolate contains a single enzyme with coumaphos hydrolytic activity. This enzyme had an apparent molecular mass of 66 kDa.

TABLE 5 Parathion hydrolytic activities of P230 cultures grown in the presence or absence of parathion to an OD at 595 nm of 0.280. Parathion hydrolytic activity (μmol/min/mg Culture protein) +parathion 3.36 ± 0.18 −parathion 3.13 ± 0.07

Example 5 Cloning the Gene Responsible for Coumaphos Hydrolytic Activity

Cloning Techniques and DNA Preparations

General cloning techniques, unless otherwise indicated, were standard and as described by Sambrook et al. (1989). Chromosomal DNA was extracted from A. radiobacter P230 according to the method of Gardiner et al. (1996). Briefly, an overnight culture (100 ml) of A. radiobacter P230 was pelleted by centrifugation at 5000 g for 20 minutes, washed twice with 10 ml ice-cold STE buffer (see above), and finally resuspended in 10 ml STE. Lysozyme (20 mg) was added and the cells incubated for 2 hours at 37° C. An equal volume of STE was added along with SDS (to a final concentration of 2% [w/v]) and RNase (to a final concentration of 20 μg/ml) and the cell lysate incubated at 42° C. for 1 hour. Proteinase K (50 μg/ml final concentration) was then added and the lysate incubated at 55° C. until the solution became translucent. An equal volume of buffer-saturated phenol/chloroform (1:1) was added, the sample thoroughly mixed and then centrifuged at 5000 g for 1 hour at 4° C. The upper aqueous layer was transferred to a clean tube using a broken pipette in order to prevent shearing of DNA. To precipitate the chromosomal DNA, 3M sodium acetate, pH5.2, (0.1 volume) and ice-cold ethanol (2.5 volumes) were added, the solution mixed gently and placed at −20° C. for 1 hour. The DNA was then removed using a “hooked” pasteur pipette. This precipitate was washed with 70% ethanol and air-dried for 5 minutes. TE buffer (pH8.0; 1 ml) was added and the DNA left to dissolve at 4° C. overnight.

Library Construction in E. coli

A partial Sau3AI digest of A. radiobacter P230 chromosomal DNA was prepared by digesting 37.5 μg of DNA with 4, 2, 1, 0.5 and 0.25 units of Sau3AI restriction endonuclease for 10 minutes at 37° C. DNA fragments in the size range of 10-12 kb were excised from a 0.7% agarose gel and extracted using a QIAGEN PCR purification/gel extraction kit, according to the manufacturer's instructions. pBluescript KS+ plasmid DNA (Stratagene), prepared using the Geneworks Ultraclean Plasmid miniprep kit, was digested with BamHI for 1 hour at 37° C. and dephosphorylated using calf intestinal alkaline phosphatase (Boehringer Mannheim). The phosphatase was then removed using the QIAquick PCR purification kit (QIAGEN). The size-fractionated P230 DNA fragments were ligated to the BamHI digested, phosphatase treated pBluescript vector, using T4 DNA ligase and the T4 ligase buffer provided by New England Biolabs. Ligations were performed for 20 hours at 4° C.

The ligated DNA was then transformed into E. coli DH10β using the revised Hanahan transformation method (Sambrook et al., 1989). The transformation mix was plated onto LB agar plates containing ampicillin (100 μg/ml), X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside; 40 μg/ml) and IPTG (isopropyl-β-D-thiogalactoside; 40 μg/ml), and incubated at 37° C. overnight. Approximately 350 white colonies were revealed.

Triparental Matings for Expression in A. tumefaciens

Since it was possible that the gene encoding the OP hydrolytic activity in A. radiobacter would not be expressed sufficiently in E. coli to allow identification on the basis of expression, the pBluescript plasmids from the E. coli library above were transferred into a closely-related strain, A. tumefaciens C58. A. tumefaciens C58 is a non-OP hydrolysing strain of Agrobacterium (Zimmerer at al., 1966). Briefly, the white colonies identified above on LB plates containing ampicillin, X-Gal and IPTG were patched onto LB plates, without any additions. Also patched onto the same plates at the same places were A. tumefaciens C58 and E. coli JM109 (Yanisch-Perron et al., 1985) containing the conjugative, cointegrative plasmid, pR751::Tn813 (Bowen and Pemberton, 1985). It was intended that the conjugative cointegrative plasmid would move into E. coli DH10β containing the pBluescript-derivatives and form a cointegrate. The cointegrates would then be transferred into A. tumefaciens C58.

The tri-parental matings were incubated overnight at 28° C. The mating mixtures were then scraped up and assayed for coumaphos hydrolytic activity in a microtitre plate. This involved resuspending the mating mix in an assay buffer containing 0.5% Triton X-100, 100 μM coumaphos and 50 mM Tris-HCl pH8.0. The microtitre plates were then incubated for 8 hours at room temperature, and the amount of fluorescence noted as described above. One mating mix (containing clone p65) demonstrated significant fluorescence.

Coumaphos Hydrolytic Activity of E. coli DH10β p65 Cell-Free Extracts

Cell-free extracts of E. coli DH10β p65, and control extracts containing the pBluescript vector alone, were prepared from cells grown to mid-log phase on LB medium containing ampicillin (100 μg/ml). The 50 ml cultures were pelleted by centrifugation at 8000 g for 15 minutes and resuspended in 2 ml 50 mM Tris-HCl pH8.0. The cells were disrupted by sonication (five 15 second bursts at 4° C.) and large cell debris or intact cells were removed by centrifugation (8000 g for 15 minutes). Aliquots (containing 15 μg protein) of the supernatants were assayed for coumaphos hydrolytic activity. The increase in fluorescence over time was measured and the amount of activity determined. It can be seen from Table 6 that cell-free extracts of E. coli DH10β containing clone p65 displayed significant coumaphos hydrolytic activity compared to that of the vector-only controls.

Localisation of the Gene Encoding the OP Hydrolytic Activity in Clone p65

Clone p65 DNA was digested to completion with HindIII and the resultant four fragments [5.5 kb (containing pBluescript vector), 4 kb, 3.5 kb and 1.4 kb] separated and subsequently excised from a 1% agarose gel. The fragments were extracted using the QIAquick PCR purification kit (QIAGEN) and ligated to HindIII digested pBluescript DNA prepared as described above. The ligation mixes were transformed into E. coli DH10β and individual clones assayed for coumaphos hydrolytic activity. Several of the clones containing the 4 kb HindIII fragment demonstrated coumaphos hydrolytic activity, depending on the orientation of the fragment in pBluescript relative to the lac promoter.

TABLE 6 Coumaphos hydrolytic activity of cell-free extracts of E. coli DH10β p65 and control extracts containing the pBluescript vector alone. Coumaphos hydrolytic activity (nmol/min/mg Strain protein) E. coli DH10β (pBluescript) 0.78 ± 0.04 E. coli DH10β (p65) 3.30 ± 0.07

Example 6 Sequence of opdA

The nucleotide sequence of the 4 kb HindIII fragment identified above was determined using primers complementary to the T3 and T7 promoters in the vector and ‘primer walking’. DNA was sequenced using the BigDye Terminator system (Applied BioSystems) on the Applied BioSystems ABI PRISM 377 automated DNA sequencer. An open reading frame (ORF) was identified in the same orientation as the lacZ promoter in clones possessing activity and in the opposite orientation in clones lacking activity. The open reading frame contains 1152 nucleotides (FIG. 2) and, when translated, would encode a protein of 384 amino acids (FIG. 3) and 41.4 kDa.

Sequence similarities were calculated using the FASTA algorithm (Pearson and Lipman, 1988). This indicated that the ORF had 88% nucleotide sequence identity to opd, a previously identified phosphotriesterase gene from Flavobacterium sp. ATCC27551 (Mulbry and Karns, 1989). Furthermore, the inferred amino acid sequence of the ORF was 90% identical to that of the Flavobacterium OPD enzyme (FIG. 4). For this reason we have named this open reading frame ‘opdA’.

Some notable differences were observed between the Flavobacterium opd sequence and that of opdA from A. radiobacter P230 (FIGS. 2 and 3). There appears to be one less amino acid in the putative signal sequence of the OpdA protein and the signal cleavage site is also different. Furthermore, a frameshift near the 3′ end of the opdA gene gives OpdA an additional 16 amino acids. This region has been sequenced multiple times to ensure that the extra base in opdA is not a sequencing error.

The native OPD enzyme is a homodimer that contains two zinc ions per monomeric subunit (Benning et al., 1995). The two His residues at positions 254 and 257 in the OPD protein sequence are located near the bimetallic active site present in each monomer and are thought to interact with active site residues and the substrate in the substrate binding pocket. Replacement of each of these His residues with Arg and Leu, respectively, resulted in enzymes possessing only two metal atoms per dimer (diSioudi et al., 1999). The OpdA protein has Arg and Tyr at the positions corresponding to His 254 and His257 in OPD (FIG. 4). It would therefore be expected that the OpdA native enzyme would contain only two metal ions per dimer rather than four, as in native OPD.

Example 7 Activity of the Purified OpdA Protein

To confirm that the open reading frame in FIG. 3 encoded the protein responsible for OP hydrolytic activity, the protein was expressed and purified as a fusion protein with maltose-binding protein.

Expression of OpdA and OPD as Fusion Proteins

The OpdA and OPD proteins were expressed in Escherichia coli using the pMAL protein fusion and purification system of New England Biolabs, which results in the expression of maltose-binding protein (MBP) fusion proteins.

To clone the opdA gene into the pMAL-c vector, the opdA gene (without the signal peptide domain) was amplified by the polymerase chain reaction (PCR) using the upstream and downstream primers, 5′GATCGTCTGCAGCCAATCGGTACAGGCGATCTG (SEQ ID NO: 11) and 5′GATCGTAAGCTTTCATCGTTCGGTATCTTGACGGGGAAT (SEQ ID NO: 12), respectively. A PstI cloning site was inserted at the start codon and a HindIII cloning site at the stop codon (underlined bases). The PCR fragment was subsequently cloned into the PstI-HindIII cloning sites of pMAL-c, to generate the recombinant plasmid, pmal-opdA.

The opd gene (Mulbry and Karns, 1989) was cloned into the pMAL-c2× vector (New England Biolabs) in a similar way. The opd gene, without the signal peptide domain, was amplified using PCR. The upstream and downstream oligonucleotide primers, 5′GATCGTGGATCCTCGATCGGCACAGGCGATCGG (SEQ ID NO: 13) and 5′GATCGTAAGCTTTCATGACGCCCGCAAGGTCGG (SEQ ID NO: 14), respectively, were designed to contain a BamHI restriction site at the opd start codon and a HindIII restriction site at the stop codon (underlined bases). The PCR fragment was subsequently cloned into the BamHI-HindIII restriction sites of pMAL-c2× to generate the recombinant plasmid, pFmal.

Purification of OpdA and OPD Proteins

Both MBP fusion proteins were expressed in E. coil DH10β cells. Optimal production of MBP fusion proteins was obtained when mid-log cells (OD₆₀₀=0.6) were induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside for 5 hours at 37° C. Harvested cells were disrupted by sonication and the soluble fraction loaded onto an amylose resin (New England Biolabs), equilibrated with 50 mM Tris-HCl pH7.5. MBP fusion proteins were eluted with 10 mM maltose in 50 mM Tris-HCl pH7.5. Fractions containing coumaphos hydrolytic activity were pooled and cleaved with Xa protease (10 μg/ml; New England Biolabs) for 5 hours. The cleaved fractions were then passaged through a DEAE sepharose ion exchange resin. Cleaved OpdA and OPD proteins did not bind to this resin and eluted with the void volume. Fractions from this sample appeared to be pure as judged by SDS-PAGE. The amount of protein in purified samples was calculated according to the method of Gill and von Hippel (1989).

Kinetic Analyses of OPD and OpdA

(i) Substrates

Kinetic parameters were determined for the hydrolysis by OpdA and OPD of the following substrates: coumaphos, parathion (O,O-diethyl p-nitrophenyl phosphorothioate; Riedel de Haan), parathion-methyl (O,O-dimethyl p-nitrophenyl phosphorothioate; Riedel de Haan), paraoxon (O,O-diethyl p-nitrophenyl phosphate; Sigma), coroxon (3-chloro-4-methyl-7-coumarinyl diethyl phosphate; Alltech), fenthion (O,O-dimethyl O-[3-methyl 4-(methylthio)phenyl]phosphorothioate; Riedel de Haan), diazinon (labelled—O,O-diethyl-O-(2-isopropyl-4-methyl-6-pyrimidinyl)-phosphorothioate; Alltech), dMUP (O,O-dimethyl 4-methyl-umbelliferyl phosphate; a gift from Alan Devonshire), chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate; Alltech) and phosmet (S-[(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)methyl O,O-dimethyl phosphorodithioate; Alltech).

(ii) Assays

All reactions contained organophosphates dissolved in methanol, except for phosmet, which was dissolved in acetone. The concentration of acetone or methanol in the reactions was constant at 5%, where appropriate. All reactions were performed in 50 mM Tris-HCl pH 8.0 at 25° C.

The initial rates of reactions of purified OPD and OpdA with coumaphos and coroxon were determined using the fluorimetric assay described above.

The initial rates of reaction of both OPD and OpdA with dMUP were determined using a fluorimetric assay to quantitate the formation of the hydrolysis product, 4-methyl umbelliferone (Roth, 1969). The fluorescence was measured using an excitation wavelength of 355 nm and an emission intensity of 460 nm.

The initial rates of reaction of both purified enzymes with parathion, parathion-methyl and paraoxon were measured spectrophotometrically by quantitating the formation of the hydrolysis product, p-nitrophenol, at 405 nm and using an extinction coefficient of 17 000 M⁻¹cm⁻¹ (Dumas et al., 1989b).

The initial rates of reaction of OpdA with fenthion were quantitated spectrophotometrically by monitoring a reduction in absorbance at 252 nm (Ibrahim and Cavagnol, 1966). A lack of hydrolysis of fenthion and phosmet by OPD was confirmed by thin layer chromatography after 24 hour incubation of the substrate with OPD.

The reaction rates of OpdA and OPD with chlorpyrifos were measured spectrophotometrically by monitoring the increase in absorbance at 276 nm (Dumas et al., 1989b).

The hydrolysis of phosmet was measured by quantitating the formation of free thiols during the course of the reaction using DTNB (Ellman's reagent; 5′5 dithio-bis-(2-nitro benzoic acid)) as has been described previously for monitoring P—S hydrolysis in organophosphates (Lai et al., 1995). This involved the addition of DTNB (80 μl of 1 mg/ml in 50 mM sodium phosphate pH7.5 and methanol, 1:1 (v/v)) to 20 μl aliquots of the reaction taken at various times.

The hydrolysis of diazinon was monitored using radiolabelled diazinon (ethyl-1-¹⁴C; 14.8 MBq/mmol) in the radiometric partition assay previously used for radiolabelled OP substrates (Campbell et al., 1998). At various times during the reaction, an aliquot (50 μl) was removed and diluted with 150 μl water. This was then extracted with 500 μl dichloromethane. The upper aqueous phase (150 μl) was removed and quantitated by liquid scintillation.

Results

Results of the kinetic analyses are given in Table 7. OpdA and OPD were able to hydrolyse the substrates coumaphos, coroxon, paraoxon, parathion, parathion-methyl, diazinon, chlorpyrifos and dMUP. OpdA had a higher k_(cat) for both parathion-methyl and dMUP, and OPD was unable to hydrdolyse either phosmet or fenthion. A lack of hydrolysis of the latter two substrates was also observed over a 24 hour period by thin layer chromatography (Munnecke and Hsieh, 1976). This involved the extraction of a 100 μl reaction containing 0.4 mM substrate with an equal volume of ethyl acetate. The upper organic phase was gently dried with a nitrogen stream, and the remaining residue was dissolved in 10 μl acetone and then applied to a neutral silica gel F₂₅₄ TLC plate (Alltech, NSW, Australia). The plate was then developed in hexane-chloroform-methanol (7:2:1) and compounds visualised by short wavelength ultra-violet light. Hydrolysis of both phosmet and fenthion were consistently observed for OpdA and no hydrolysis was seen for OPD.

In summary, several differences in substrate specificity between OpdA and OPD were observed. OpdA hydrolysed fenthion and phosmet whereas OPD did not. Furthermore, there was a significant difference between OpdA and OPD in the k_(cat) values for dimethyl OPs, with OpdA possessing a higher k_(cat) for methyl-parathion and dMUP than OPD. We would also expect OpdA, like OPD (Dumas et al., 1989a; Yang et al, 1995), to hydrolyse OP nerve agents.

As discussed above, the two His residues at positions 254 and 257 in the OPD protein sequence are located near the bimetallic active site present in each monomer and are thought to interact with active site residues and the substrate in the substrate binding pocket (Benning et al., 1995). Replacement of each of these His residues with Arg and Leu, respectively, resulted in enzymes with only one metal ion per monomer, increased catalytic activity for larger substrates such as demeton, and decreased activity for smaller

TABLE 7 Kinetic parameters of purified OpdA and OPD enzymes for various OP substrates. K_(m) (μM) k_(cat) (min⁻¹) Substrate/Structure OpdA OPD OpdA OPD

8.3 ± 1.8 21.4 ± 6.0  12.4 ± 0.6  14.1 ± 2.6 

15.9 ± 1.9  25.3 ± 1.3  22.7 ± 0.1  39.5 ± 5.3 

242 ± 61  225 ± 14  33.5 ± 0.5  46.0 ± 0.4 

92.6 ± 6.4  50.6 ± 12.2 21.9 ± 2.0  23.5 ± 0.2 

61.2 ± 2.3  32.9 ± 1.7  94.2 ± 0.8  5.46 ± 0.05

208.3 ± 13.2  — 0.100 ± 0.002 —

148.6 ± 17.2  — 1.63 ± 0.01 — . . . /cont.

51.9 ± 4.5  54.2 ± 5.4  65.2 ± 6.7  56.5 ± 2.9 

32.6 ± 8.1  47.2 ± 3.0  0.525 ± 0.005 0.90 ± 0.01

66.0 ± 9.1  46.7 ± 2.8  81.7 ± 9.1  20.5 ± 2.3  substates like paraoxon (diSioudi et al., 1999). It was postulated that changes in the number of bound metal ions may enhance structural flexibility and improve access of larger substrates to the active site, while simultaneously decreasing activity for smaller substrates. The OpdA protein has Arg and Tyr at the positions corresponding to His 254 and His257 in OPD (FIG. 4). It is therefore surprising that OpdA possessed a higher k_(cat) for methyl-parathion than OPD, yet its k_(cat) for the larger ethyl-parathion substrate was similar to that of OPD. Similar results were obtained for the coumaphos/dMUP substrate pair. Clearly, differences between the OpdA and OPD amino acid sequences other than those at residues 253/254 and 256/257 affect catalytic activity.

Example 8 Identification of OpdA Mutants with Altered Specificity

The plasmid pmal-opdA was transformed into the E. coli mutator strain XL1-red. The plasmid was propagated in this strain for 120 generations, with plasmid extractions occurring after every 24 generations. These plasmids were then transformed into E. coli DH1013 and the transformation mix diluted to 50 ml in LB containing ampicillin.

When the culture reached an OD₅₉₅ of 0.3, fusion protein expression was induced with 0.1 mM IPTG, and induction allowed to occur for 5 hours. The culture was then pelleted by centrifugation, resuspended in 2 ml of sterile 50 mM Tris-HCl pH7.5 with the addition of malathion to a final concentration of 440 μM. This assay mixture was left for 1 hour and the hydrolysis of malathion detected using Ellman's reagent (DTNB) (Lai et al., 1995).

Pools containing activity were then diluted and plated onto LB plates with ampicillin. Individual colonies were then selected and tested for malathion and dimethoate hydrolytic activity as described above. Two colonies (designated pmal-opdA1 and pmal-opdA2) were selected and examined further.

The sequences of the two mutants were examined and compared with that of wild-type OpdA. OpdA1 contained 4 mutations (P42S, P134S, A170S and S237G) (SEQ ID NO: 3) and OpdA2 contained one mutation (A119D) (SEQ ID NO: 4). The numbering system is based on OpdA numbering of amino acid residues, taking into account the signal sequence. To correlate the numbering with OPD, add one to each number.

OpdA and the two mutants OpdA1 and OpdA2 were purified after expression in the plasmid pCY76. The genes were amplified by PCR using the primers pETopdA5 (5′GATCGTGAATTC CATATGCCAATCGGTACA, with EcoRI site underlined and NdeI double underlined) (SEQ ID NO: 15) and pETopdA3 (5′GATCGTGGATCCTCATCGTTCGGTATCTTG, with BamHI site underlined) (SEQ ID NO:16). PCR fragments were digested with EcoRI and BamHI and ligated with similarly-digested pBluescript. Sequence of the fragments were confirmed in this vector. The pBS-derivatives were then digested with Ndel-BamHI and ligated with Ndel-BglII-digested pCY76. Positive clones were grown in 500 ml of LB. After the cultures had grown for 24 hours, they were pelleted by centrifugation at 7000 g, 15 minutes at 4° C. The pellets were resuspended in 4 ml 50 mM Tis-HCl pH7.5 and broken by sonication (Harcourt et al., 2002). Cell-free extracts were then charged onto a DEAE sepharose column that was pre-equilibrated with 50 mM Tris-HCl pH7.5. OpdA and variants did not bind to this column and the eluant was collected and placed on a heparin sepharose column pre-equilibrated with 50 mM Tris-HCl pH7.5 (Pharmacia). OpdA and variants were bound by this column and eluted with 50 mM Tris-HCl pH7.5/0.1 M NaCl. After this column step, OpdA was judged to be pure by SDS-PAGE. The kinetics of the proteins were examined against the aliphatic OPs, dimethoate, malathion, malaoxon and DFP (diisopropyl fluorophosphate) (Table 8). Both mutants were active against dimethoate, malathion and malaoxon, whereas the wild-type OpdA was not. Furthermore, the mutants had increased activity for DFP compared to that of wild-type OpdA.

TABLE 8 The kinetic parameters of purified OpdA and the mutants, OpdA1 and OpdA2, for various OP substrates. K_(m) (μM) k_(cat) (min⁻¹) Substrate OpdA OpdA1 OpdA2 OpdA OpdA1 OpdA2

2.3 ± 0.4 18.1 ± 0.6  9.6 ± 5.6 1.36 ± 0.04 45.9 ± 0.9  27.5 ± 0.7 

nd¹ 78.9 ± 17.7 14.3 ± 4.2  nd 1.22 ± 0.07 1.4 ± 0.1

nd 33.3 ± 14.2 40.9 ± 7.2  nd 1.21 ± 0.07 1.43 ± 0.07

nd 159.2 ± 28.2  45.7 ± 8.9  nd 1.98 ± 0.05 1.84 ± 0.06 ¹nd = not detected

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

All publications discussed above are incorporated herein in their entirety.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

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The invention claimed is:
 1. A host cell comprising a recombinant polynucleotide which encodes a polypeptide comprising amino acids whose sequence is at least 95% identical to the sequence set forth as SEQ ID NO:2 or a composition comprising the polypeptide recovered from the host cell.
 2. The host cell or composition of claim 1, wherein the polypeptide comprises amino acids whose sequence is at least 97% identical to the sequence set forth as SEQ ID NO:2.
 3. The host cell or composition of claim 1, wherein the polypeptide is a substitution mutant of the polypeptide comprising amino acids whose sequence is set forth as SEQ ID NO:2.
 4. The host cell or composition of claim 1, wherein the polynucleotide comprises nucleotides whose sequence is at least 95% identical to the sequence set forth as SEQ ID NO:6.
 5. The host cell or composition of claim 1, wherein the polynucleotide is comprised in a vector.
 6. The host cell or composition of claim 5, wherein the vector is a plasmid vector.
 7. The host cell or composition of claim 5, wherein the vector is integrated into one or more host cell chromosomes.
 8. The host cell or composition of claim 1, wherein the host cell is a bacterial cell or a yeast cell.
 9. The host cell or composition of claim 8, wherein the bacterial cell is Salmonella sp., Escherichia sp., Bacillus sp., or Listeria sp.
 10. The host cell or composition of claim 8, wherein the bacterial cell is Escherichia coli.
 11. The host cell or composition of claim 2, wherein the host cell is a bacterial cell or a yeast cell.
 12. The host cell or composition of claim 11, wherein the bacterial cell is Salmonella sp., Escherichia sp., Bacillus sp., or Listeria sp.
 13. The host cell or composition of claim 11, wherein the bacterial cell is Escherichia coli.
 14. The host cell or composition of claim 3, wherein the host cell is a bacterial cell or a yeast cell.
 15. The host cell or composition of claim 14, wherein the bacterial cell is Salmonella sp., Escherichia sp., Bacillus sp., or Listeria sp.
 16. The host cell or composition of claim 14, wherein the bacterial cell is Escherichia coli.
 17. A composition comprising the host cell of claim 1 and one or more acceptable carriers.
 18. A composition comprising the host cell of claim 14 and one or more acceptable carriers.
 19. A composition comprising the host cell of claim 15 and one or more acceptable carriers.
 20. A composition comprising the host cell of claim 16 and one or more acceptable carriers.
 21. A process for preparing a polypeptide comprising amino acids whose sequence is at least 95% identical to the sequence set forth as SEQ ID NO:2, the process comprising cultivating the host cell of claim 1 under conditions which allow production of the polypeptide, and extracting the polypeptide. 